Pregnancy-specific responses to COVID-19 revealed by high-throughput proteomics of human plasma

Background Pregnant women are at greater risk of adverse outcomes, including mortality, as well as obstetrical complications resulting from COVID-19. However, pregnancy-specific changes that underlie such worsened outcomes remain unclear. Methods Plasma samples were collected from pregnant women and non-pregnant individuals (male and female) with (n = 72 pregnant, 52 non-pregnant) and without (n = 29 pregnant, 41 non-pregnant) COVID-19. COVID-19 patients were grouped as asymptomatic, mild, moderate, severe, or critically ill according to NIH classifications. Proteomic profiling of 7,288 analytes corresponding to 6,596 unique protein targets was performed using the SOMAmer platform. Results Herein, we profile the plasma proteome of pregnant and non-pregnant COVID-19 patients and controls and show alterations that display a dose-response relationship with disease severity; yet, such proteomic perturbations are dampened during pregnancy. In both pregnant and non-pregnant state, the proteome response induced by COVID-19 shows enrichment of mediators implicated in cytokine storm, endothelial dysfunction, and angiogenesis. Shared and pregnancy-specific proteomic changes are identified: pregnant women display a tailored response that may protect the conceptus from heightened inflammation, while non-pregnant individuals display a stronger response to repel infection. Furthermore, the plasma proteome can accurately identify COVID-19 patients, even when asymptomatic or with mild symptoms. Conclusion This study represents the most comprehensive characterization of the plasma proteome of pregnant and non-pregnant COVID-19 patients. Our findings emphasize the distinct immune modulation between the non-pregnant and pregnant states, providing insight into the pathogenesis of COVID-19 as well as a potential explanation for the more severe outcomes observed in pregnant women.

Pregnant COVID-19 patients are at increased risk of experiencing complications and severe outcomes compared to the general population. However, the reasons for this heightened risk are still unclear. We measured the proteins present in the blood of pregnant and non-pregnant patients with COVID-19 and compared these to healthy individuals. We found that some COVID-19associated proteins were present at lower levels in pregnant women, which could help to protect the fetus from harmful inflammation, the body's natural response to infection. While some proteins affected by COVID-19 are shared between pregnant and non-pregnant patients, others were distinctly affected only in pregnant women, providing a potential explanation for the more severe outcomes in this group. C oronavirus disease 2019 (COVID- 19) represents an ongoing threat to people around the world 1,2 . As of October 2022, over 600 million people have been infected with SARS-CoV-2, the virus responsible for COVID-19, and the death toll has neared 6.5 million 1 . A growing body of evidence has indicated that pregnant women are at an increased risk of adverse outcomes resulting from COVID-19, ranging from greater rates of admission to the intensive care unit and need for mechanical ventilation to higher risk of death compared to nonpregnant women [3][4][5] . Moreover, pregnant women with COVID- 19 have also been shown to experience more obstetrical complications such as preeclampsia 6,7 , preterm birth 6,7 , and stillbirth 8 . Thus, COVID-19 during pregnancy not only adversely affects the mother, but can also negatively affect short-and long-term quality of life for the offspring by causing respiratory distress syndrome, increasing neonatal intensive care unit admission, and impairing cognitive development, among others [9][10][11][12][13][14][15][16][17] . Hence, there is an urgent need to understand the pregnancy-driven biological pathways, including immune responses, underlying the increased susceptibility to severe COVID-19 and obstetrical disease.
Upon the onset of the COVID-19 pandemic, multiple investigations have sought to uncover the effects of SARS-CoV-2 infection on maternal, fetal/placental, and neonatal physiology, including the immune response [18][19][20][21][22][23][24][25][26][27][28][29][30][31] . Indeed, we and others have characterized the changes in systemic parameters such as cellular immune responses, virus-specific immunoglobulins, and inflammatory mediators in the maternal peripheral blood and/or cord blood to generate a profile of the maternal-fetal immune responses against SARS-CoV-2 infection 25,[32][33][34][35] . In particular, comparative studies of pregnant and non-pregnant COVID-19 patients showed that pregnant women were more likely to experience a cytokine storm 36 characterized by specific mediators [37][38][39] , together with elevated neutrophil counts 37,38 and lymphopenia 39 , providing insight into the potential mechanisms underlying the increased susceptibility to severe COVID-19 during pregnancy. The recent utilization of longitudinal and multi-omics approaches has allowed for the identification of specific processes contributing to COVID-19 progression and severity in the general population [40][41][42][43] . Thus, to further understand the consequences of COVID-19 in pregnant women, the application of high-throughput omics platforms can facilitate the identification of relevant molecules and biological pathways implicated in this disease. Indeed, a recent study profiled over 1400 proteins in maternal peripheral blood and cord blood and indicated that pregnant women with severe COVID-19 display increased inflammatory and anti-viral signaling compared to asymptomatic pregnant women, while their offspring displayed elevated cytokines associated with T-cell responses and/or inflammasome activation 44 . However, the proteomic dysregulation that distinguishes pregnant from non-pregnant COVID-19 patients has not been elucidated.
Aptamer-based technologies that allow the identification and monitoring of over 1000 potential target proteins have been utilized to profile the human proteome during normal pregnancy and/or its complications in the maternal plasma [45][46][47][48][49] and amniotic fluid 50 . Yet, the much-expanded version (4.1) of the SOMAScan platform, which allows measuring of over 7000 analytes, had not been utilized to study pathology in obstetrics.
In this study, we classify pregnant and non-pregnant women according to COVID-19 status and severity and perform proteomic profiling using the high-throughput SOMAScan platform to determine the differentially affected proteins. We show that COVID-19 drives changes in the plasma proteome in a doseresponse relation with disease severity. Importantly, the response to COVID-19 is dampened during pregnancy, regardless of disease severity. Distinct and overlapping proteomic changes are identified in pregnant and non-pregnant COVID-19 patients: pregnant women display a tailored proteomic response, potentially to protect the conceptus from inflammation, while nonpregnant women display a stronger response that may help fight off infection. Moreover, the stereotypical proteomic response induced by COVID-19 in the pregnant and non-pregnant states shows enrichment of mediators implicated in a cytokine storm, endothelial dysfunction, and angiogenesis; yet, such a response is dampened during pregnancy. Finally, we apply machine learning to demonstrate that the plasma proteome can be used to discriminate COVID-19 patients from controls, even those who are asymptomatic or have mild symptoms.

Methods
Study design. The study involved profiling 7288 proteomic targets in plasma samples collected from pregnant women (n = 101) and from non-pregnant individuals (n = 93, including both men and women) of Hispanic ethnicity (Colombia). Hispanic refers to a person of South or Central American, or other Spanish culture or origin, regardless of race, as defined by the NIH (https://grants. nih.gov/grants/guide/notice-files/not-od-15-089.html). Pregnant patients were enrolled at admission to the labor and delivery unit or at the time of attending the clinic due to obstetrical indications or clinical deterioration warranting inpatient management. While the term "pregnant women" is used to describe this cohort, we note that we did not ask whether any of these patients identified as transgender or nonbinary. Variables considered to be indicative of clinical deterioration were the following: SOFA score at patient admission; presence of dyspnea; vital signs: heart rate >90, respiratory rate >20, median blood pressure; altered mental status; ICU admission; white blood cell count; D-dimer value; troponin values; OIT requirement; admission to in care diagnosis; admission to obstetric in care diagnosis; Glasgow coma scale; abnormal diagnostic images; abnormal electrocardiogram findings and echocardiogram; platelet values; hemoglobin values; serum lactate values; arterial blood gas values; serum electrolyte values; procalcitonin values; positive blood cultures; and clotting times. Moreover, for maternal clinical deterioration, we considered the following maternal (ICU admission; mechanical ventilation; non-severe preeclampsia; severe preeclampsia; gestational hypertension; eclampsia; pulmonary embolism; abruption; prelabor rupture of membranes; preterm labor; postpartum hemorrhage; maternal mortality; antenatal corticosteroid therapy) and perinatal (abnormal antepartum fetal monitoring; fetal growth restriction; acute respiratory distress syndrome; neonatal sepsis; IVH; prematurity; congenital malformation; NICU admission; perinatal mortality) outcomes.
All patients were screened for COVID-19 according to standard clinical care. Patients with suspected COVID-19 were triaged and admitted to the respiratory ward, where they underwent PCR testing to confirm the diagnosis of COVID-19. Depending on disease severity, patients were transported to the pulmonary ICU for treatment as well as clinical/paraclinical management. Of all controls (patients without  enrolled in the study, we retained those who had samples collected within the same gestational age window as cases. Nonpregnant patients were enrolled at the time of admission for any medical indication, and all were tested for COVID-19 as described above. For non-pregnant women with COVID-19, diagnoses at admission were recorded as viral pneumonia, ARDS, septic shock, or other. For non-pregnant women without COVID-19, diagnoses at admission were recorded as coronary disease, kidney failure, or other. All analyses accounted for the age and sex of patients as well as the presence of chronic hypertension or high-risk pathology. All patients provided written informed consent, and the use of biological specimens and clinical data for research purposes was approved by the Biomedical Research Ethics Committee of the Fundacion Valle del Lili (Protocol No. 1611), Cali, Colombia. Patients diagnosed with COVID-19 were grouped as asymptomatic, mild, moderate, severe, or critically ill according to NIH classification 51 . Blood samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA), and plasma was separated by centrifugation (1600 × g for 10 min at 4°C). Plasma samples were immediately stored at −80°C until proteomic analysis.
Plasma proteomics. Maternal plasma protein abundance was determined using the SOMAmer (Slow Off-rate Modified Aptamers) platform, which enables the multiplexed quantification of 7288 analytes corresponding to 6596 unique protein targets [52][53][54] . We present these results at the level of analytes, also referred to as proteins. Experiments were run in randomized batches by blinded laboratory personnel. Briefly, plasma samples were diluted and then incubated with the respective SOMAmer mixes preimmobilized onto streptavidin-coated beads, which were then washed to remove unbound proteins and other matrix constituents. Bound proteins were tagged using an NHS-biotin reagent. After labeling, the beads were exposed to an anionic competitor solution that prevents non-specific interactions from reforming after disruption. Pure cognate-SOMAmer complexes and unbound SOMAmer reagents were released from the streptavidin beads using ultraviolet light. The photo-cleavage eluate was separated from the beads and then incubated with a second streptavidin-coated bead that binds the biotin-labeled proteins and the biotin-labeled protein-SOMAmer complexes. Subsequently, free SOMAmer reagents were removed by washing. Finally, protein-bound SOMAmer reagents were released from their cognate proteins using denaturing conditions. SOMAmer reagents were quantified by hybridization to custom DNA microarrays to detect the cyanine-3 signal 52-54 . Proteomics profiling was performed by Somalogic, Inc. (Boulder, CO, USA).

Statistics and reproducibility
Demographic and clinical characteristics. These data were summarized using numbers and percentages for categorical variables or medians and interquartile range (IQR) for continuous variables. Differences between cases and controls were assessed using Fisher's exact test for categorical data and the Wilcoxon test for continuous data. All statistical tests were two-tailed and significance was inferred based on p < 0.05.
Principal component analysis. Protein abundances expressed as relative fluorescence units (RFU) were log 2 -transformed to improve normality. The function prcomp in the R statistical language and environment (www.r-project.org) was used to calculate principal components (PC). The top three PC were tested for associations with COVID-19 and pregnancy status using linear models with interaction terms. The dose-response relationship between a given PC and disease severity was assessed using a linear model in which the response variable was the PC and the explanatory variable was an ordered factor with six levels ordered in the sequence: Control, Asymptomatic, Mild, Moderate, Severe, and Critical. This analysis included also pregnancy status, age, and sex of participants as possible confounding variables. All statistical tests were two-tailed and significance was inferred based on p < 0.05. The top 2% of proteins with the largest absolute loading values on each of the first three principal components were interpreted via biological process enrichment analysis as described below in Gene ontology enrichment analysis.
Differential abundance analysis. The proteomic data preprocessing, including an adaptive normalization by maximum likelihood (ANML) step and a calibration step, were performed by SomaLogic, Inc. The goal of these steps was to make data comparable across samples by calculating plate-specific and analytespecific scale factors. After log (base 2) transformation, data were compared between pooled COVID-19 cases and controls or compared separately between each disease severity group against controls. When analyzing data from pregnant women, maternal age, body mass index (BMI), and linear and quadratic terms of gestational age were included as co-variates. Analysis of data from non-pregnant subjects included adjustment for age, BMI, and sex of the participant. Models were fit using the limma package 55,56 in R. Protein abundance was considered to have changed significantly with COVID-19 if the fold change was >1.25 and false discovery rate (FDR) 57 adjusted p value (q value) was <0.1. Spearman correlation coefficients and significance p-values were calculated to determine the similarity of log 2 fold changes in protein abundance obtained for different COVID-19 severity groups against controls, both within and between pregnant and non-pregnant subjects. Proteins with opposite dysregulation due to COVID-19 between pregnant and non-pregnant groups were defined as proteins being either a) significantly changed with COVID-19 in pregnant women (q < 0.1, fold change >1.25) but with opposite direction of change in non-pregnant individuals (p < 0.05), or b) significantly changed with COVID-19 in nonpregnant individuals (q < 0.1, fold change >1.25) but with opposite direction of change in pregnant women (p < 0.05).
Gene ontology enrichment analysis. Proteins were mapped using the Entrez gene database 58 identifiers based on SomaLogic, Inc. protein annotation followed by Gene Ontology 59 . Biological processes over-represented among a given protein set were identified using Fisher's exact tests. Gene ontology terms with three or more hits and an adjusted enrichment q value <0.1 were considered as significantly enriched. The MSigDB collection 60 of curated canonical pathways (C2 collection) was also analyzed. Enrichment tests were performed using the GOStats package 61 in Bioconductor enrichment analyses. Biological processes overrepresented among a given protein set were identified using Fisher's exact tests. Gene ontology terms with three or more hits and an adjusted enrichment q value <0.1 were considered as significantly enriched. The MSigDB collection 60 of curated canonical pathways (C2 collection) was also analyzed. Enrichment tests were performed using the GOStats package 61 in Bioconductor 62 .
Predictive model development. To assess the value of plasma proteomic data to discriminate between COVID-19 and controls, we have developed random forest models using up to 50 proteins. The proteins were selected based on their importance to the accuracy of the models using the randomForest function in R. The protein selection and random forest model fitting steps were evaluated using leave-one-out cross-validation (LOOCV), and receiver operating characteristic curves were derived using the pROC package in R.
Reporting summary. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Results
Characteristics of the study population Pregnant individuals. Plasma samples were collected from 101 pregnant women (23.2-39.3 weeks of gestation), including those diagnosed with COVID-19 at the time of admission (n = 72) and those who tested negative for SARS-CoV-2 during prenatal care visits (hereafter referred to as pregnant controls; n = 29) (Fig. 1a, b and Table 1). Parameters such as maternal age, BMI, parity, presence of labor at the time of sampling, frequency of chronic hypertension, and diagnosis of preeclampsia in the current pregnancy were comparable between the pregnant COVID-19 and control groups (Table 1). Gestational age at delivery was similar between groups; yet, sampling of COVID-19 cases was performed~5 weeks earlier in gestation than in controls [median weeks (IQR) controls: 36.1 (32.6-37.5) vs. COVID-19: 31.3 (28.1-35.6), p < 0.05] (Table 1), which was considered in the data analysis. Among the pregnant COVID-19 cases, 6 (8%) were asymptomatic, 20 (28%) were mild, 13 (18%) were moderate, 12 (17%) were severe, and 21 (29%) were critically ill according to NIH classification 51 .
COVID-19 drives shared and unique changes in the plasma proteome in pregnant and non-pregnant individuals that follow a dose response with disease severity. Over 7000 protein analytes were determined using the SOMAScan v4.1 platform in cases and controls to characterize the plasma proteome responses induced by COVID-19 according to its severity in pregnant and non-pregnant individuals (Fig. 1a). Uniform Manifold Approximation and Projection (UMAP) plots of the proteomic profiles illustrate that patients are clustered according to COVID-19 status and severity in both pregnant and non-pregnant (Supplementary Fig. 1a, b) individuals. It is worth mentioning that, in non-pregnant individuals, the plasma proteome was heavily modulated by COVID-19 status, regardless of sex (Supplementary Fig. 2). We performed an unsupervised projection of highdimensional proteomic profiles of all controls and COVID-19 patients onto the first three principal components (PC), which can be understood as meta-proteins that are uncorrelated with each other (Fig. 1c, d). Notably, pregnancy status represented a source of variability in the proteome, as PC2 values (18% of variance explained) perfectly discriminated between pregnant and non-pregnant individuals (p < 0.001, Fig. 1c). The proteins with the top 2% highest loadings in PC2 were enriched for biological processes such as pregnancy, reproductive process, and gonadotropin secretion (q < 0.1, odds ratios >3 for all). Yet, the host response to COVID-19 represented the primary source of variability in the proteome, as PC1 and PC3 (PC1, 27% of variance explained; PC3, 6% of variance explained) were significantly different between COVID-19 cases and controls, regardless of pregnancy status (p < 0.001 for both, Fig. 1d). Proteins with the highest contribution to PC1 and PC3 were enriched for biological processes such as exocytic process (PC1), anti-viral innate immune response, antimicrobial humoral response, and positive regulation of type I interferon production (PC3) (q < 0.1, odds ratios >4 for all). The proteomic changes with COVID-19 were larger for non-pregnant than for pregnant women based on both PC1 and PC3 (interaction p < 0.005) (Fig. 1d), which is partly explained by the greater proportions of severe and critically ill cases in the non-pregnant than in the pregnant population. Moreover, we observed a dose-response relationship between PC3 and disease severity, regardless of pregnancy status (p < 0.001 for both linear and quadratic trends, Fig. 1e). Together, these data provide an overview of the plasma proteome in pregnant and non-pregnant individuals infected with SARS-CoV-2, and suggest dramatic changes with infection in a dose-response relationship with disease severity. In addition, these data hint that the host response to SARS-CoV-2 includes shared and unique processes between pregnant and non-pregnant individuals, which we further explore below.
The plasma proteome response to COVID-19 follows a doseresponse relationship with disease severity in pregnant and non-pregnant individuals, yet such a response is dampened in pregnancy. Pregnant women have been reported to display heightened susceptibility to severe COVID-19 [3][4][5] . Therefore, we first explored the differential effects of COVID-19 on the maternal proteome compared to gestational age-matched control pregnancies according to disease severity (Fig. 2a). When comparing pregnant COVID-19 cases to controls after adjustment for maternal age, BMI, and gestational age at sampling, we identified 68, 81, 242, 144, and 1072 differentially abundant proteins in asymptomatic, mild, moderate, severe, and critically ill cases, respectively ( Fig. 2b-f). Given that both disease severity and sample size may affect the number of differentially abundant proteins in specific groups, we next used the protein changes between critically ill patients and controls (1072 proteins) as a reference to compare with the changes observed in the less severe COVID-19 groups (Fig. 2g). The log 2transformed fold change of protein abundance between COVID-19 subgroups (i.e., asymptomatic, mild, moderate, and severe) and controls were more attenuated than those between critically ill patients and controls (regression slopes <1.0) (Fig. 2g). Yet, the magnitude of correlation and the correlation slope followed a doseresponse relationship with disease severity, and even asymptomatic patients showed plasma proteomic changes that were significantly correlated to those observed in critically ill patients (r = 0.34 for Asymptomatic vs. Control; r = 0.72 for Mild vs. Control; r = 0.87 for Moderate vs. Control; r = 0.88 for Severe vs. Control; p < 0.001 for all) (Fig. 2g).
We then performed the same analysis of differential protein abundance in non-pregnant patients (Fig. 3a), and identified 21,1961, and 2966 differentially abundant proteins in moderate, severe, and critically ill cases, respectively ( Fig. 3b-d), after adjusting for relevant covariates. Similar to the analysis in pregnant women, the log 2 -transformed fold changes of protein abundance between COVID-19 subgroups and controls were more attenuated than those found between critically ill cases and controls, and followed a dose-response with disease severity (r = 0.84 for Moderate vs. Controls; r = 0.94 for Severe vs. Controls; p < 0.001 for both) (Fig. 3e).
To contrast the magnitude of COVID-19-driven changes in the proteome between pregnant and non-pregnant patients, we then performed correlation analysis based on a core set of 486 proteins with significant and consistent changes in both pregnant and nonpregnant patients ( Fig. 4a and Supplementary Data 1). These proteins with shared perturbation in pregnant and non-pregnant women with COVID-19 can be grouped into four main clusters enriched for distinct biological processes ( Fig. 4b and Supplementary Data 2). Clusters 1 and 2 were predominantly increased in COVID-19 cases, regardless of sex or pregnancy status, while Clusters 3 and 4 were primarily decreased ( Fig. 4b and Supplementary Data 2). By comparing the magnitude of changes between the pregnant and non-pregnant groups, we showed that the magnitude of changes for this set of core proteins was diminished during pregnancy for the same disease severity group, as indicated by the regression line slopes below 1.0 ( Fig. 4c-e, p < 0.05 for all).
Together, these results demonstrate that there is a perturbation of the plasma proteome in both pregnant and non-pregnant women with COVID-19, and that the magnitude of such changes increases with COVID-19 severity. However, relative to the plasma proteome perturbations observed in non-pregnant individuals, the magnitude of changes with COVID-19 in the pregnant state is attenuated, suggesting a dampened response.
Shared and distinct changes in the plasma proteome of pregnant and non-pregnant women with COVID-19. We then sought to further unravel pregnancy-driven differences in the plasma proteomic response to COVID-19 as well as changes that are shared between pregnant and non-pregnant states. First, we identified all proteins that were differentially abundant with COVID-19, which resulted in 708 differentially abundant proteins for pregnant women ( Next, we explored the biological processes that were enriched among the entire set of differentially abundant proteins for pregnant (708 proteins) and non-pregnant (2605 proteins) COVID-19 patients to characterize the differences in host response (Fig. 5c-f). As expected, enriched biological processes in pregnant women with COVID-19 were fewer than those in non-pregnant patients, given the dampened protein response (Fig. 5c and Supplementary Data 4-6). Consistent with such an observation, pregnant COVID-19 patients showed enrichment of processes related to extracellular matrix, defense response, and immune response (Fig. 5d), whereas those enriched in nonpregnant individuals included protein localization and transport, peptide biosynthesis, and translation ( Fig. 5e and Supplementary Data 4,5). Shared processes were characterized by cell adhesion and immune responses as well as response to wounding and blood coagulation (Fig. 5f). We summarized the biological processes perturbed with COVID-19 in pregnant and non-pregnant patients according to disease severity (Supplementary Data 6).
In addition to biological processes, we also evaluated the enrichment of pathways and gene sets derived from the C2 collection of the MSigDB database, which includes canonical pathways and experimental gene sets such as those associated with disease and viral infection ( Fig. 5g and Supplementary Data 7-9). Similar to biological processes, pathways enriched in pregnant women with COVID-19 included terms related to extracellular matrix; yet, pathways associated with viral infection or anti-viral defenses were also observed ( Fig. 5h and Supplementary Data 7). Enriched pathways in non-pregnant COVID-19 patients included terms related to platelet activation, VEGF, and PDGF (Fig. 5i), while shared pathways included virus-and cancer-related terms ( Fig. 5j and Supplementary Data 8). The shared C2 pathways perturbed with COVID-19 between pregnant and non-pregnant patients were also summarized while considering disease severity (Supplementary Data 9).
Together, these data further demonstrate that, although there is a set of common responses to COVID-19 in both pregnant and nonpregnant state, pregnancy-specific changes are detectable in the maternal circulation. These data put forward evidence for a working hypothesis that pregnancy tailors the immune response against pathogens 29,63,64 threatening the successful completion of gestation.
COVID-19 drives distinct and shared angiogenic and inflammatory proteomic changes in pregnant and non-pregnant individuals. Given our finding that pregnancy modifies the proteomic response to COVID-19, we further investigated whether any proteins were dysregulated with COVID-19 in opposite directions between pregnant and non-pregnant patients (see Methods). This analysis identified a core set of 33 proteins with opposing direction of change (Fig. 6a) and included proteins related to angiogenesis and wound healing as well as alarmins, cytokines, and growth factors (Supplementary Data 10). Proteins that decreased with COVID-19 in pregnancy but were increased in non-pregnant cases included vascular endothelial growth factor receptor 1 (VEGF-sR1 or sFLT-1) and angiotensinogen (AGT); yet, this could potentially be explained by their already elevated baseline among pregnant patients (Fig. 6b, c and Supplementary Data 10). Consistent with these findings, proteins that underwent pregnancy-specific regulation with COVID-19 were enriched for biological processes and pathways related to vasodilation, angiogenesis, and regulation of inflammatory response (Supplementary Data 11). A previous report indicated that COVID-19 during pregnancy is characterized by a profile of proteomic factors that is distinct from but overlaps with that observed in preeclampsia 6 , an obstetric syndrome characterized by intravascular inflammation 65 . Therefore, we further evaluated changes in angiogenic or endothelial factors between pregnant and non-pregnant COVID-19  patients. Several factors such as soluble TNF receptor II (TNFRSF1B) and von Willebrand factor (VWF) were found to increase with COVID-19 regardless of pregnancy status (Fig. 6d,  e). Notably, neutrophil elastase (ELANE), a neutrophil degranulation factor 66 as well as a component of neutrophil extracellular traps (NETs) 67 , was elevated in both pregnant and non-pregnant COVID-19 cases (Fig. 6f), as was histone H3.1 (H3C1), another NET component (Fig. 6g). These results provide insight into the unique biological processes in pregnant and non-pregnant individuals: while non-pregnant individuals exhibit increased abundance of angiogenic and inflammatory proteins in the circulation, the proteome of pregnant women hints at a systemic inflammatory response and no increase in anti-angiogenic sFLT-1, which is already elevated in the pregnant state.
Pregnant women with COVID-19 display a dampened systemic cytokine response. COVID-19 is characterized by a cytokine storm, components of which can display a dose-response with disease severity 41 . Therefore, we next focused on the protein expression changes of specific inflammatory mediators (Fig. 7a). The classical inflammatory cytokines IL-6, IL-1β, and IL-18 were increased in COVID-19 cases compared to controls for both pregnant and non-pregnant patients; yet, the latter two did not reach significance in pregnant women (IL-1β, p = 0.074; IL-18, p = 0.052), likely due to the dampened proteomic response (Fig. 7b-d). Similarly, TNF and IL-17A were upregulated with COVID-19 in non-pregnant patients and only showed a slight tendency to increase during pregnancy (Fig. 7e, f). The alarmin IL-1α was found to be downregulated only in pregnant COVID-19 cases, although a tendency towards the same reduction was observed in non-pregnant patients (Fig. 7g). By contrast, IFNγ was reduced with COVID-19 in non-pregnant individuals but not pregnant patients (Fig. 7h). The anti-inflammatory cytokine IL-10 was downregulated in pregnant and non-pregnant COVID-19 cases (Fig. 7i), whereas TGFβ1 was upregulated in both groups (Fig. 7j). Several chemokines were also found to exhibit differential regulation with COVID-19 in the pregnant and non-pregnant states: CXCL10 and CCL22 were consistently increased or diminished, respectively, in both non-pregnant and pregnant cases; yet, CCL1 was reduced and CXCL13 was increased only in non-pregnant COVID-19 patients, although data from pregnant patients showed similar tendencies (Fig. 7k-n). These findings suggest that COVID-19 induces a cytokine storm in the circulation of both pregnant and non-pregnant individuals; yet, pregnant women display a dampened soluble immune response. The plasma proteome can discriminate COVID-19 cases from uninfected controls, even when mild or asymptomatic. Last, we evaluated the ability of the proteomic profiles to discriminate between COVID-19 cases and controls, regardless of pregnancy status. For this purpose, we developed random forest models that included up to 50 proteins and evaluated their accuracy via leave-one-out cross-validation (LOOCV). The resulting proteomics model was able to accurately discriminate COVID-19 cases from controls, in the absence of any other inputs (Fig. 8a). The area under the Receiver Operating Characteristic curve (AUC) was 0.978 for the full analysis set, 0.974 for pregnant women, and 0.985 for non-pregnant individuals (Fig. 8a). The relative ARTICLE COMMUNICATIONS MEDICINE | https://doi.org/10.1038/s43856-023-00268-y importance of the proteomic predictors in the random forest model is displayed in Fig. 8b and includes several of the proteins with differential abundance as reported in Supplementary Data 1 and 3. When classification models were derived separately based on disease severity, the accuracy to distinguish most severe cases (severe or critical COVID-19) from controls was higher (AUC = 0.99) than the one obtained for discriminating between controls and moderate cases (AUC = 0.94) (Fig. 8c). Of interest, similarly high accuracy was obtained also for distinguishing asymptomatic or mild cases from uninfected controls (AUC = 0.95) (Fig. 8c). ISG15, MX1, ZBP1 and IFNL1 were the top four proteins most contributing to the accuracy of random forest models for discriminating all COVID-19 cases from controls, and these proteins were also among the top ones for prediction of severe and critical COVID-19 (Fig. 8d), moderate COVID-19 ( Supplementary Fig. 3), and for mild or asymptomatic cases ( Supplementary Fig. 4). Together, these data suggest that a shared proteomic signature can discriminate between COVID-19 patients and healthy individuals regardless of pregnancy status, and that disease severity is a driver of classification accuracy.

Discussion
In this study, we utilized the SOMAScan v4.1 platform to profile over 7000 protein targets in the peripheral blood of pregnant women and non-pregnant individuals diagnosed with COVID-19, and found that this disease drives changes in their plasma proteomes in a dose-response relation with disease severity. Importantly, we showed that the response to COVID-19 is dampened during pregnancy, regardless of disease severity. Distinct and overlapping proteomic changes were identified in pregnant and non-pregnant COVID-19 patients: pregnant women displayed a tailored proteomic response, potentially to protect the conceptus from the deleterious effects of inflammation, while non-pregnant women displayed a stronger response to fight off infection. The stereotypical proteomic response induced by COVID-19 in the pregnant and non-pregnant state showed enrichment of mediators implicated in cytokine storm, endothelial dysfunction, and angiogenesis, yet was dampened during pregnancy. Last, we utilized machine learning to demonstrate that the plasma proteome can be used to discriminate COVID-19 patients from controls, even those who were asymptomatic or had mild symptoms. The proteomic dysregulations after COVID-19 revealed in our current study are suggestive of a dampened systemic immune response in pregnant women compared to non-pregnant individuals, both in terms of the number of proteins affected and magnitude of changes for proteins implicated in the pregnant and nonpregnant states. This phenomenon could be secondary to physiological changes that occur during pregnancy, such as the reversible thymic involution 68-70 that impacts T-cell development 71,72 , or could be a primary outcome intended to prevent aberrant immune activation that could threaten pregnancy 73,74 . Immune suppression was originally considered to be a requirement for successful pregnancy, given the immunological puzzle of the mother displaying tolerance towards the semi-allograft fetus for 40 weeks 75 . Rather than complete inertness or unresponsiveness, as proposed by Peter Medawar 75 , pregnancy has since been shown to be a state of selective immune tolerance [76][77][78][79][80][81][82][83][84][85][86][87] , mediated by homeostatic cells such as regulatory T cells (Tregs) [76][77][78][79][80][81]84,85,[87][88][89][90][91][92][93][94][95][96][97] and macrophages 92,[98][99][100][101][102][103][104][105][106] . This concept is further supported by studies of women with autoimmune diseases such as systemic lupus erythematosus (SLE), in whom such pregnancy-specific immune adaptations can fail to occur 107,108 , resulting in pregnancy complications 108,109 . Pertinent to our current findings, it was suggested that the suppression of key immune pathways such as IFN could underlie the higher risk of severe viral infection during pregnancy 108 . Given the high energy demands of fighting off infection, it is possible that the diversion of maternal resources to effectively clear the virus while avoiding a potentially harmful inflammatory response may result in a transient period of fetal "neglect", which may contribute to the observed worsened outcomes in pregnant COVID-19 patients. Indeed, past and present viral pandemics have provided a large body of evidence showing that specific viruses, such as pandemic influenza viruses, Dengue virus, and coronaviruses, can result in disproportionately high rates of adverse outcomes in pregnant women 110 .
Peripheral T and B cells show decreased numbers, greater activation-induced proliferation, and altered phenotypes during pregnancy 64,111 , and such alterations can be further exacerbated by the lymphopenia that characterizes viral infections such as SARS-CoV-2 35,41,42,112 . Moreover, given the demonstrated relationship between pathological maternal T-cell activation and pregnancy complications such as preterm labor 73,74 , it is imperative that maternal adaptive immunity remain under strict control until normal parturition at term [113][114][115][116] . Consistently, we recently undertook an ex vivo evaluation of peripheral cellular immune responses against SARS-CoV-2 particles and proteins in pregnant and non-pregnant women 29 . We demonstrated a pregnancy-specific reduction of unswitched memory-like and transitional-like B-cell subsets 29 , which is in line with a prior study showing that such reduction of B cells is associated with COVID-19 severity 117 . Thus, given such deficiencies in peripheral adaptive immunity, pregnant women infected with SARS-CoV-2 may rely more heavily on monocytes, which are also potent contributors to anti-viral host defense 118 . Consistently, monocytes undergo substantial expansion and differentiation in patients with severe COVID-19 [119][120][121] , and we have shown that monocytes from pregnant women appear to undergo accelerated transition and activation in response to SARS-CoV-2 exposure 29 , which is in line with a previous report 37 .
Notably, we found that the cytokine profile of peripheral leukocytes was also impacted by pregnancy, as the release of IFN-β  and IL-8 in response to SARS-CoV-2 was diminished compared to non-pregnant women 29 . The abovementioned studies, together with our current results, point to a specific dampening of the maternal proteomic response to COVID-19 to protect the fetus from heightened inflammation that could jeopardize pregnancy. This may not be the only mechanism protecting the fetus, as the placenta has also been shown to play a critical role in anti-SARS-CoV-2 defenses 35,122 . The incidence of vertical transmission of SARS-CoV-2 has been shown to be rare, which may be due in part to the minimal co-expression of the canonical viral cell entry mediators ACE2 and TMPRSS2 in this organ 18 . Moreover, the placenta exhibits strong anti-viral properties 123,124 , and in women with COVID-19 the placental anti-viral response was shown to include the activation of leukocytes such as T cells, NK cells, and macrophages together with elevated expression of genes related to immune and cytokine signaling, even in the absence of detectable placental infection 35,122 . Thus, the diminished maternal systemic response to SARS-CoV-2 infection may be partially offset by the protective functions of the placenta, thereby preventing a cytokine storm that could damage the fetus while still ensuring a barrier to prevent viral transmission.
Herein, we found that pregnant and non-pregnant patients infected with SARS-CoV-2 exhibit a perturbed proteomic profile characterized by the enhanced release of cytokines and other mediators associated with inflammation, endothelial dysfunction, and angiogenesis. A hallmark of severe COVID-19 is the systemic inflammatory response that includes the exacerbated release of pro-inflammatory immune mediators, termed a cytokine storm [125][126][127][128][129] . Multiple cytokines involved in this response have been proposed as biomarkers of severity and prognosis for COVID-19 130 . Indeed, the peripheral blood concentration of cytokines, including IL-6, is highly correlated with mortality in patients with COVID-19 130,131 , hinting at a key role for IL-6 in the pathophysiology of severe disease. In fact, it has been proposed that IL-6 acts as an amplifier of the inflammatory response triggered by SARS-CoV-2 by activating the NF-κB and STAT3 pathways in non-immune cells such as the vascular endothelium 132 . This concept is in line with the clinical findings showing that the cytokine storm can lead to generalized endothelial dysfunction 127,133 , as was initially suspected early in the pandemic given the rapid emergence of cardiovascular complications in COVID-19 patients 134,135 . The vascular endothelium is an organ with multiple endocrine, paracrine, and autocrine functions, which are required for vascular homeostasis and regulation of vascular tone 136,137 . Therefore, any disruption in these functions can induce vasoconstriction that can progress to ischemia, inflammation, edema, and culminate in a pro-coagulant state 138 .
In addition to the indirect induction of endothelial dysfunction due to the host inflammatory response 139,140 , SARS-CoV-2 can also directly interact with the vascular endothelium, as evidenced by viral inclusion structures observed in vascular endothelial cells at multiple body sites in deceased COVID-19 patients 140,141 . SARS-CoV-2 binds to the ACE2 receptor to enter cells, which can impair the activity of the enzyme ACE2 to neutralize angiotensin vasopressors 133,142 . Such impaired ACE2 activity can activate the kallikrein-bradykinin pathway that results in increased vascular permeability 133,143 . Moreover, the activation of innate immune cells induces the release of toxic mediators such as reactive oxygen species (ROS) and vasoactive substances that can lead to Fig. 8 The plasma proteome allows for the identification of COVID-19 patients and can distinguish mild and severe diseases. a Receiver operating characteristic (ROC) curves for discrimination of all COVID-19 cases from all controls (black curve), only pregnant COVID-19 cases from pregnant controls (red curve), and only non-pregnant COVID-19 cases from non-pregnant controls (blue curve). b Bar plot displaying the relative importance of the top 50 proteomic predictors for identifying all COVID-19 cases. c ROC curves for discrimination of severe/critical cases from controls (red curve), moderate cases from controls (yellow curve), and asymptomatic/mild cases from controls (blue curve). Data from both pregnant and non-pregnant cases are included in this analysis. d Bar plot displaying the relative importance of the top 50 proteomic predictors for distinguishing severe/critical COVID-19 cases from controls.
inter-endothelial gaps, thereby further enhancing endothelial permeability 133 . The activation of endothelial cells leads to the production of multiple pro-coagulant factors, such as P-selectin, fibrinogen, and Von Willebrand factor (VWF), which initiates the coagulation cascade 133,139 . These processes can also lead to platelet aggregation and the release of other factors such as VEGF, which upregulates the endothelial cell production of tissue factor, the primary stimulator of the coagulation cascade 133,144 , ultimately leading to a pro-thrombotic state. Consistently, herein we showed that, while non-pregnant patients with COVID-19 exhibit angiogenic and inflammatory circulatory profiles, the proteome of pregnant women is characterized by a systemic inflammation without dysregulating the anti-angiogenic factor sFLT-1, which is already elevated in pregnant controls.
Initial investigations of pregnant women infected with SARS-CoV-2 had revealed the development of a preeclampsia-like syndrome 145,146 . Later evidence supported COVID-19 as a risk factor for preeclampsia 7,147 and indicated a dose-response relationship with disease severity 6 ; however, the mechanisms and causality of such an association are still poorly understood 65,147,148 . Our current findings revealed that some proteins implicated in inflammatory and angiogenic processes were perturbed in patients with COVID-19, regardless of pregnancy status; yet, specific proteins were only modified by SARS-CoV-2 infection in pregnancy. As preeclampsia is a primarily systemic endothelial-inflammatory obstetrical disease 65,[149][150][151][152][153] , our findings support the fact that some perturbed pathways may be shared between COVID-19 and preeclampsia. A previous study compared circulating biomarkers in pregnant women with COVID-19 and those of women with preeclampsia, and demonstrated that these conditions display distinct biomarker profiles 154 . The partial overlap between these two disease states may be driven by the placental inflammatory response induced by maternal SARS-CoV-2 infection, even in asymptomatic pregnant women 35,122 . Such inflammation can affect the fetus even in the absence of vertical transmission 35 ; therefore, it is imperative to follow and evaluate these infants for eventual adverse outcomes, as suggested by recent evidence demonstrating neurodevelopmental sequelae at one year of life in children exposed to SARS-CoV-2 in utero 16,155 .
The establishment of biomarkers that allow for the classification and monitoring of COVID-19 outcomes is essential to guide patient management, particularly during pregnancy. In the current study, we demonstrated that the systemic proteome can be utilized to distinguish COVID-19 patients and controls, in the absence of any other patient risk factors. Of importance, the plasma proteome was able to discriminate asymptomatic cases and those with mild symptoms from controls with high accuracy. The top proteomic contributors in our discriminatory model included interferon-induced GTP-binding protein Mx1 (MX1 or MXA), interferon-stimulated gene 15 (ISG15), Z-DNA-binding protein 1 (ZBP1), and interferon lambda 1 (IFNL1), also referred to as IL-29. Given their responsiveness to IFN signaling, ISG15 and its downstream targets, including MX1 156 , have been implicated as key participants during SARS-CoV-2 infection by using in vitro and in vivo models [157][158][159] as well as samples from COVID-19 patients [160][161][162] . Similarly, the induction of IFNL1 by SARS-CoV-2 infection was previously reported in vitro 163 . Analysis of serum samples derived from pregnant women with COVID-19 using a different proteomic platform indicated the specific upregulation of IFNL1 and its receptor, IFNLR1, in severe/critical patients compared to mild/moderate or asymptomatic 44 , which conflicts with earlier findings that IFN responses are delayed and impaired in non-pregnant COVID-19 patients 164 . Here, we observed consistent upregulation of IFNL1 in pregnant and non-pregnant COVID-19 patients compared to controls; yet, consistent with the abovementioned study 44 , this interferon most strongly contributed to the discrimination of severe/critical cases from controls, suggesting it is most greatly impacted in such patients. ZBP1 is part of the innate immune response 165 that is induced by IFN signaling [166][167][168] and mediates PANoptosis (pyroptosis, necroptosis, and apoptosis) as part of host defense against pathogens such as influenza 169 . Importantly, ZBP1 has also been shown to be activated by SARS-CoV-2 infection 166,[170][171][172] . Given its function as an inducer of PANoptosis, the IFN-mediated upregulation of ZBP1 represents an obstacle to the proposed use of IFNs as a potential therapy for COVID-19 173 . Further studies exploring such therapy may consider the inhibition of ZBP1 in conjunction with IFN therapy to help prevent the associated tissue damage and lethality 173 .
Our current findings are in line with a prior multi-omics investigation that evaluated 1400 plasma proteins together with single-cell immune features for the classification of non-pregnant COVID-19 patients 174 . In the latter study, such integrated modeling showed value for the distinction of mild, moderate, and severe COVID-19 cases, and identified specific immune features that showed dose-response changes with disease 174 . The use of specific inflammatory mediators in the circulation to characterize COVID-19 was evaluated since the onset of the pandemic, with elevated levels of cytokines (such as IL-6), chemokines, and interferons being reported in cases of severe COVID-19 175,176 and high systemic levels of IL-6, IL-8, and TNF at the time of hospitalization showing use as biomarkers of disease severity and mortality 130 . In-depth investigations have used longitudinal profiling of COVID-19 patients to identify multiple immune signatures that correlated with different disease trajectories 41 , or utilized proteomic determinations and machine learning to identify 11 host proteins and biomarker combinations that could distinguish and predict COVID-19 outcomes 177 . Interestingly, the presence of neutralizing immunoglobulin G (IgG) autoantibodies against type I interferons has also been shown to represent a likely indicator of severe disease in COVID-19 patients, given that such autoantibodies were absent in most of the individuals with asymptomatic or mild SARS-CoV-2 infection 178 . Together with our current data, these observations point to the value of identifying specific proteomic changes that can serve as biomarkers of COVID-19 severity, particularly during the vulnerable period of pregnancy. It is worth mentioning that the proteomic approach used herein was previously validated by our group 179 , indicating that our determinations are robust and can serve for future studies using targeted assays.
The current study has some limitations. Information regarding the interval from infection or symptom onset to sampling collection was not available, and thus any potential differences in the kinetics of the proteomic response to COVID-19 between pregnant and non-pregnant patients could not be considered. In addition, while the non-pregnant controls included in the study were matched by age and comorbidity with non-pregnant cases, we acknowledge that the inclusion of a healthy control group representing a non-perturbed proteomic state would be ideal. It should also be noted that specifically profiling the proteome to diagnose COVID-19 may not be practical; yet, the signature of COVID-19 described herein could be used to generate risk scores for potential COVID-19 at the time of sample collection in studies focused on other outcomes. Indeed, blood proteomic Soma Signal tests have already been proposed as patient-specific health indicators for cardiovascular events 180 , liver fat, kidney health, percentage body fat, cardiopulmonary fitness, and diabetes, among others 181 . The cardiovascular events risk score was shown to be informative of subsequent COVID-19 severity and mortality 180 , supporting the implementation of the COVID-19 signature proposed herein as a signal test to provide a more comprehensive characterization of the health status of subjects participating in studies with other primary endpoints.
Collectively, the study herein represents the most comprehensive characterization of the plasma proteome of pregnant and non-pregnant individuals diagnosed with COVID-19. The findings reported herein emphasize the distinct immune modulation between the non-pregnant and pregnant states, providing insight into the pathogenesis of COVID-19 as well as a potential explanation for the more severe outcomes observed in pregnant women. Importantly, the unique proteomic profiles observed in pregnant women suggest that the preeclampsia-like disease in this population may differ in pathogenesis from the canonical pathways implicated in the obstetrical syndrome of preeclampsia. Yet, further investigation is required to decipher the unique molecular mechanisms whereby SARS-CoV-2 infection induces a maternal cytokine storm and, more importantly, its effects on the offspring.

Data availability
The majority of the data generated in this study are included in the manuscript and/or in the Supplementary Materials. Proteomic data generated in this study are available at the Gene Expression Omnibus (accession number GSE207015). All software and R packages used herein are detailed in the Methods.